The invention relates to the biodegradation of phosphonate products. In particular, this invention is a biodegradation process for the organophosphonate product of Sarin (O-isopropyl methylphosphonofluoridate) caustic hydrolysis, namely, isopropylmethylphosphonate (IMPA). This process provides a feasible biodegradation demilitarization alternative to Sarin incineration. Public opposition of nerve agent incineration is widespread, and alternative methods are sought to help the U.S. Army meet the 2007 demilitarization deadline imposed by the Chemical Weapons Convention.
In view of the public outcry against the incineration of stockpiles of chemical agents, the U.S. Army is seeking alternative chemical agent demilitarization technologies. Microbial biodegradation is one promising alternative the Army is considering for this purpose. Recently, microbial biodegradation of hydrolyzed mustard (bis-2-chlorethyl sulfide) was sanctioned as the most promising alternative technology suitable for the Army""s demilitarization goals for that chemical agent. See Irvine, D. A., J. P. Earley, D. P. Cassidy and S. P. Harvey, xe2x80x9cBiodegradation of Sulfur Mustard Hydrolysate in the Sequencing Batch Reactor,xe2x80x9d Water Sci. and Tech. 35: 67-74 (1996), incorporated herein by reference in its entirety.
As other chemical warfare agents have different structures, the biodegradation of these materials and/or their neutralization products were pursued. The organophosphorus nerve agents VX (O-ethyl-S-2(diisopropylaminoethyl)methylphosphonothioate) and Sarin (O-isopropylmethylphosphofluoridate) represent a large portion of the Army""s stockpile. Equimolar mixtures of VX and water undergo a slow (30-50 days at room temperature) auto-catalytic reaction resulting in cleavage of the P-S bond 
to produce ethylmethylphosphonate (EMPA) and DIAESH (Diisopropylaminoethylmercaptan).
Caustic hydrolysis of Sarin also produces an alkyl phosphonate: 
specifically, isopropylmethylphosphonate (IMPA) and sodium fluoride.
As is seen, the phosphonate products of chemical hydrolysis are similar for these two agents: ethylmethylphosphonate (EMPA) is the byproduct of VX hydrolysis and isopropylmethylphosphonate (IMPA) is the byproduct of the hydrolysis of Sarin. In the past, biodegradation of these materials was accomplished using strategies similar to that for mustardxe2x80x94sequencing batch reactors. The reactors in a sequencing batch reactor system operate through a cycle of four discrete periods. The periods are fill, react, settle and draw. Waste is introduced into the reactor during fill. In such cases, the byproduct phosphonates were used as the sole phosphorus source for growth. Supplementary glucose was supplied simultaneously with the hydrolyzed agent. Although EMPA biodegradation proceeded well for the VX hydrolysate using sequencing batch reactors (DeFrank, J. J., I. J. Fry, J. P. Earley and R. L. Irvine, Biodegradation Studies with Water-Hydrolyzed Nerve Agent VX. Proceedings of the 20th Army Science Conference, p. 555-559 (1996), incorporated herein by reference in its entirety), poor results were exhibited for IMPA biodegradation when sludge sequencing batch reactors were employed for the Sarin hydrolysate (DeFrank, J. J., I. J. Fry, C. M. Frost and J. P. Earley, Sequencing Batch Reactor Biodegradation of Water-Hydrolyzed Sarin, Proceedings of the 1996 ERDEC Scientific Conference on Chemical and Biological Defense Research, p. 361-367 (1996), incorporated herein by reference in its entirety). Clearly, a better approach was needed to effectively degrade the IMPA needed for Sarin demilitarization purposes.
Prior work on alkylphosphonate biodegradation was reported by several investigators (Wanner, B. L, Phosphate-Regulated Genes for the Utilization of Phosphonates in Members of the Family Enterobacteiaceae, In: Phosphate in Microorganisms: Cellular and Molecular Biology, A. Torriani-Gorini, E. Yagil and S. Silver eds. ASM Press, Washington, D.C., pp. 215-221 (1994), incorporated herein by reference in its entirety). However, almost all of these reports focused on the mono-substituted phosphonates, such as methylphosphonate (MPA), ethylphosphonate (EPA), or xcex1-aminoethylphosphonate (AEPN). The enzyme responsible for MPA biodegradation is C-P lyase. The reaction catalyzed by this enzyme is: 
C-P lyase is inhibited by low levels of phosphate (Daughton, C. G., A. M. Cook and M. Alexander, Bacterial Conversion of Alkylphosphonates to Natural Products via Carbon-Phosphorus Bond Cleavage, J. Agric. Food Chem. 27: 1375-1382 (1979)).
The biochemistry of MPA biodegradation has been well characterized in Enterobacter, Salmonella and E. coli (Wanner, B. L, Phosphate-Regulated Genes for the Utilization of Phosphonates in Members of the Family Enterobacteriacca, In: Phosphate in Microorganisms: Cellular and Molecular Biology, A. Torriani-Gorini, E. Yagil and S. Silver eds. ASM Press, Washington, D.C., pp. 215-221 (1994), incorporated herein by reference in its entirety). Several genes for MPA uptake and the biodegradation pathway were cloned and expressed in E. coli (Wanner, B. L. and J. A. Boline, Mapping and Molecular Cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli. J. Bacteriol. 172: 1186-1196(1990), incorporated herein by reference in its entirety). IMPA biodegradation was reported in intracellular extracts of Pseudomonas testosteroni (Daughton, C. G., A. M. Cook and M. Alexander, Bacterial Conversion of Alkylphosphonates to Natural Products via Carbon-Phosphorus Bond Cleavage, J. Agric. Food Chem. 27: 1375-1382 (1979)). This organism could use several disubstituted alkylphosphonates as its sole phosphorus source but not as a sole carbon source.
Thus, an object of this invention is to provide a consortium of microorganisms which use disubstituted phosphonates as a sole carbon and phosphorus source, thereby effectively degrading said phosphonates.
The present invention is a defined aerobic bacterial consortium and process that degrades the organophosphonate product of Sarin (O-isopropyl methylphosphofluoridate) hydrolysis, namely isopropylmethylphosphonic acid (IMPA). The hydrolyzed Sarin (GBH) stock solutions that were the feed source for this invention contained 4% hydrolyzed Sarin (40 g/l) in sodium hydroxide; hence a feed of 80 ml/l GBH is actually 3.2 g/l IMPA. Initial concentrations of alkylphosphonates of from about 2.1 to 4.2 g/l can be used.
The invention degrades IMPA to methyl phosphonic acid (MPA) in the presence of equimolar fluoride ion. Isopropanol released from this reaction supplies the sole carbon source for the culture. Furthermore, the consortium degrades MPA to liberate methane and inorganic phosphate. The consortium utilizes the phosphate produced from this reaction as a sole phosphorus source. Another compound metabolized in an analogous manner by the invention is ethylmethylphosphonic acid (EMPA), one of the hydrolysis products of VX (O-ethyl-S-2(diisopropylaminoethyl) methylphosphonothioate). This was observed during bibdegradation studies of hydrolyzed Sarin (GBH), as EMPA is a minor contaminant of this preparation. The biodegradation of combined organophosphonates (EMPA, IMPA and MPA) is a characteristic of the present invention.
Thus, the invention is a defined consortium of several bacteria capable of biodegrading combined organophosphonates. These organisms have been identified or characterized and their role in hydrolyzed Sarin biodegradation defined. None of the organisms identified in this consortium have any prior published description of phosphonate biodegradation. The ability of this consortium to utilize hydrolyzed Sarin as a sole carbon and sole phosphorus source is a novel ability not previously reported for a bacterial isolate or a defined consortium.
The invention employs a process involving two bioreactors. The process comprises contacting an aqueous alkylphosphonate solution and the GB2 consortium in a first bioreactor, wherein the alkylphosphonate solution provides the sole nutritional sources of carbon and phosphorus to the bacteria under aerobic conditions; transferring the culture to a second bioreactor once the enzymatic conversion of the alkylphosphonate to MPA is complete; diluting the culture with GBMH medium; and adding a carbon source in a concentration sufficient to allow the culture to grow under aerobic conditions and thereby converting the MPA to phosphate which is utilized for growth and energy. At this point the process is complete, i.e., all of the alkylphosphonates are mineralized meeting international treaty requirements, and the contents of the second reactor can be discarded.
This invention plays a key role in the bioprocess demilitarization of the nerve agent Sarin. Microbial degradation of waste chemicals is an environmentally-compatible, relatively low cost treatment technology that has a proven track record and enjoys wide public acceptance. This invention should find applications in the biodegradation of both U.S. and foreign chemical agent stockpiles.